scholarly journals Characterization of the Caulobacter crescentus Holdfast Polysaccharide Biosynthesis Pathway Reveals Significant Redundancy in the Initiating Glycosyltransferase and Polymerase Steps

2008 ◽  
Vol 190 (21) ◽  
pp. 7219-7231 ◽  
Author(s):  
Evelyn Toh ◽  
Harry D. Kurtz ◽  
Yves V. Brun
Keyword(s):  
Caulobacter Crescentus ◽  
Repeat Unit ◽  
Deletion Mutants ◽  
Biosynthesis Pathway ◽  
Surface Adhesion ◽  
Wild Type ◽  
Chitin Deacetylase ◽  
Gene Encoding ◽  
Polysaccharide Biosynthesis

ABSTRACT Caulobacter crescentus cells adhere to surfaces by using an extremely strong polar adhesin called the holdfast. The polysaccharide component of the holdfast is comprised in part of oligomers of N-acetylglucosamine. The genes involved in the export of the holdfast polysaccharide and the anchoring of the holdfast to the cell were previously discovered. In this study, we identified a cluster of polysaccharide biosynthesis genes (hfsEFGH) directly adjacent to the holdfast polysaccharide export genes. Sequence analysis indicated that these genes are involved in the biosynthesis of the minimum repeat unit of the holdfast polysaccharide. HfsE is predicted to be a UDP-sugar lipid-carrier transferase, the glycosyltransferase that catalyzes the first step in polysaccharide biosynthesis. HfsF is predicted to be a flippase, HfsG is a glycosyltransferase, and HfsH is similar to a polysaccharide (chitin) deacetylase. In-frame hfsG and hfsH deletion mutants resulted in severe deficiencies both in surface adhesion and in binding to the holdfast-specific lectin wheat germ agglutinin. In contrast, hfsE and hfsF mutants exhibited nearly wild-type levels of adhesion and holdfast synthesis. We identified three paralogs to hfsE, two of which are redundant to hfsE for holdfast synthesis. We also identified a redundant paralog to the hfsC gene, encoding the putative polysaccharide polymerase, and present evidence that the hfsE and hfsC paralogs, together with the hfs genes, are absolutely required for proper holdfast synthesis.

scholarly journals Genetic Characterization of the Carotenoid Biosynthetic Pathway in Methylobacterium extorquens AM1 and Isolation of a Colorless Mutant

2003 ◽  
Vol 69 (12) ◽  
pp. 7563-7566 ◽  
Author(s):  
Stephen J. Van Dien ◽  
Christopher J. Marx ◽  
Brooke N. O'Brien ◽  
Mary E. Lidstrom
Keyword(s):  
Biosynthetic Pathway ◽  
Genetic Characterization ◽  
Carotenoid Biosynthesis ◽  
Biosynthesis Pathway ◽  
Wild Type ◽  
Methylobacterium Extorquens ◽  
Growth Properties ◽  
Carotenoid Biosynthesis Pathway ◽  
Free Strain

ABSTRACT Genomic searches were used to reconstruct the putative carotenoid biosynthesis pathway in the pink-pigmented facultative methylotroph Methylobacterium extorquens AM1. Four genes for putative phytoene desaturases were identified. A colorless mutant was obtained by transposon mutagenesis, and the insertion was shown to be in one of the putative phytoene desaturase genes. Mutations in the other three did not affect color. The tetracycline marker was removed from the original transposon mutant, resulting in a pigment-free strain with wild-type growth properties useful as a tool for future experiments.

Molecular characterization of three mutations in katG affecting the activity of hydroperoxidase I of Escherichia coli

1990 ◽  
Vol 68 (7-8) ◽  
pp. 1037-1044 ◽  
Author(s):  
Peter C. Loewen ◽  
Jacek Switala ◽  
Mark Smolenski ◽  
Barbara L. Triggs-Raine
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Polymerase Chain Reaction Amplification ◽  
Protein A ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Gene Encoding ◽  
Reaction Amplification ◽  
Mutant Genes

Hydroperoxidase I (HPI) of Escherichia coli is a bifunctional enzyme exhibiting both catalase and peroxidase activities. Mutants lacking appreciable HPI have been generated using nitrosoguanidine and the gene encoding HPI, katG, has been cloned from three of these mutants using either classical probing methods or polymerase chain reaction amplification. The mutant genes were sequenced and the changes from wild-type sequence identified. Two mutants contained G to A changes in the coding strand, resulting in glycine to aspartate changes at residues 119 (katG15) and 314 (katG16) in the deduced amino acid sequence of the protein. A third mutant contained a C to T change resulting in a leucine to phenylalanine change at residue 139 (katG14). The Phe139-, Asp119-, and Asp314-containing mutants exhibited 13, < 1, and 18%, respectively, of the wild-type catalase specific activity and 43, 4, and 45% of the wild-type peroxidase specific activity. All mutant enzymes bound less protoheme IX than the wild-type enzyme. The sensitivities of the mutant enzymes to the inhibitors hydroxylamine, azide, and cyanide and the activators imidazole and Tris were similar to those of the wild-type enzyme. The mutant enzymes were more sensitive to high temperature and to β-mercaptoethanol than the wild-type enzyme. The pH profiles of the mutant catalases were unchanged from the wild-type enzyme.Key words: catalase, hydroperoxidase I, mutants, sequence analysis.

Characterization of yeast V-ATPase mutants lacking Vph1p or Stv1p and the effect on endocytosis

2002 ◽  
Vol 205 (9) ◽  
pp. 1209-1219 ◽  
Author(s):  
Natalie Perzov ◽  
Vered Padler-Karavani ◽  
Hannah Nelson ◽  
Nathan Nelson
Keyword(s):  
Sucrose Gradient ◽  
Ph Sensor ◽  
Wild Type ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Vacuolar Acidification ◽  
Atpase Subunits ◽  
Immunological Assays ◽  
Gradient Fractionation

SUMMARYSubunit a of V-ATPase in the yeast Saccharomyces cerevisiae, in contrast to its other subunits, is encoded by two genes VPH1 and STV1. While disruption of any other gene encoding the V-ATPase subunits results in growth arrest at pH 7.5, null mutants of Vph1p or Stv1p can grow at this pH. We used a polyclonal antibody to yeast Stv1p and a commercially available monoclonal antibody to Vph1p for analysis of yeast membranes by sucrose gradient fractionation, and two different vital dyes to characterize the phenotype of vph1 ▵ and stv1 ▵mutants as compared to the double mutant and the wild-type cells. Immunological assays of sucrose gradient fractions revealed that the amount of Stv1p was elevated in the vph1 ▵ strain, and that vacuoles purified by this method with no detectable endosomal contamination contain an assembled V-ATPase complex, but with much lower activity than the wild type. These results suggest that Stv1p compensates for the loss of Vph1p in the vph1 ▵ strain. LysoSensor Green DND-189 was used as a pH sensor to demonstrate unexpected changes in vacuolar acidification in stv1▵ as the Vph1p-containing V-ATPase complex is commonly considered to acidify the vacuoles. In the vph1 ▵ strain, the dye revealed slight but definite acidification of the vacuole as well. The lipophilic dye FM4-64 was used as an endocytic marker. We show that the null V-ATPase mutants, as well as the vph1 ▵ one, markedly slow down endocytosis of the dye.

scholarly journals Characterization of the SOS Regulon of Caulobacter crescentus

2007 ◽  
Vol 190 (4) ◽  
pp. 1209-1218 ◽  
Author(s):  
Raquel Paes da Rocha ◽  
Apuã César de Miranda Paquola ◽  
Marilis do Valle Marques ◽  
Carlos Frederico Martins Menck ◽  
Rodrigo S. Galhardo
Keyword(s):  
Dna Damage ◽  
Caulobacter Crescentus ◽  
Bacterial Species ◽  
Direct Repeat ◽  
Site Directed Mutagenesis ◽  
Dependent Manner ◽  
Wild Type ◽  
Promoter Regions ◽  
Potential Promoter

ABSTRACT The SOS regulon is a paradigm of bacterial responses to DNA damage. A wide variety of bacterial species possess homologs of lexA and recA, the central players in the regulation of the SOS circuit. Nevertheless, the genes actually regulated by the SOS have been determined only experimentally in a few bacterial species. In this work, we describe 37 genes regulated in a LexA-dependent manner in the alphaproteobacterium Caulobacter crescentus. In agreement with previous results, we have found that the direct repeat GTTCN7GTTC is the SOS operator of C. crescentus, which was confirmed by site-directed mutagenesis studies of the imuA promoter. Several potential promoter regions containing the SOS operator were identified in the genome, and the expression of the corresponding genes was analyzed for both the wild type and the lexA strain, demonstrating that the vast majority of these genes are indeed SOS regulated. Interestingly, many of these genes encode proteins with unknown functions, revealing the potential of this approach for the discovery of novel genes involved in cellular responses to DNA damage in prokaryotes, and illustrating the diversity of SOS-regulated genes among different bacterial species.

scholarly journals The capsular polysaccharide biosynthesis of Streptococcus pneumoniae serotype 8: functional identification of the glycosyltransferase WciS (Cap8H)

2005 ◽  
Vol 389 (1) ◽  
pp. 63-72 ◽  
Author(s):  
Nehmé SAKSOUK ◽  
Ludovic PELOSI ◽  
Pierre COLIN-MOREL ◽  
Manel BOUMEDIENNE ◽  
Patricia L. ABDIAN ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Biochemical Characterization ◽  
Repeat Unit ◽  
Sugar Residue ◽  
Functional Identification ◽  
Polysaccharide Biosynthesis ◽  
Sugar Addition ◽  
Galactose Residue

CPS (capsular polysaccharide) is a major virulence factor in Streptococcus pneumoniae. Biosynthesis of CPS RU (repeat unit) proceeds by sequential transfer of sugar residues from the appropriate sugar donor to an activated lipid carrier by committed GTs (glycosyltransferases). While the nucleotide sequence of many cps loci is already known, the real substrate specificity of the hypothetical GTs, as well as the sequence of sugar addition is unclear. In the present paper, we report the biochemical characterization of one α-galactosyltransferase, WciS (Cap8H), a member of GT family 4. This enzyme is implicated in the tetrasaccharide RU biosynthetic pathway of Strep. pneumoniae CPS 8 ([→4)-α-D-Glcp-(1→4)-α-D-Galp-(1→4)-β-D-GlcAp-(1→4)-β-D-Glcp-(1→]n). Expression of WciS–His6 in Escherichia coli BL21 (DE3) strains or BL21 (DE3)/ΔgalU strain resulted in synthesis of a 39 kDa membrane-associated protein identified by N-terminal sequencing and recognized by anti-His6-tag antibody. This protein was capable of adding a galactose residue cellobiuronic acid [β-D-GlcAp-(1→4)-D-Glcp]-pyrophosphate-polyprenol from UDP-Gal. The newly added galactose residue is removed by α-galactosidase, indicating that WciS is a retaining GT. Our results suggest that WciS catalyses the addition of the third sugar residue of the CPS 8 RU. The recombinant WciS–His6 was solubilized and purified as a soluble multimer, opening the way for structural studies.

scholarly journals Transcriptome-wide Identification and Quantification of Caffeoylquinic Acid Biosynthesis Pathway and Prediction of their Putative BAHDs Gene Complex in A. spathulifolius

2021 ◽  
Author(s):  
SeonJoo Park ◽  
Jean Claude Sivagami ◽  
Sunmi Park
Keyword(s):  
Phenylpropanoid Pathway ◽  
Biosynthesis Pathway ◽  
Gene Complex ◽  
Biotic And Abiotic Stress ◽  
Acid Biosynthesis ◽  
Gene Encoding ◽  
Caffeoylquinic Acid ◽  
Key Enzyme ◽  
By Products

The phenylpropanoid pathway is a major secondary metabolite pathway that helps plants overcome biotic and abiotic stress and produces various by-products that promote human health. Its byproduct, chloroquinic acid (CQA), is a soluble phenolic compound present in many angiosperms. Hy-droxycinnamate-CoA shikimate/quinate transferase(BAHDs superfamily enzyme) is a significant en-zyme that plays a role in accumulating CQA biosynthesis. This study analyzed transcriptome-wide identification of the phenylpropanoid to chloroquinic acid biosynthesis candidate genes in A. spathulifolius flowers and leaves. Transcriptomic analyses of the flowers and leaves showed a differential expression of the PPP and CQA biosynthesis regulated unigenes. An analysis of PPP captive unigenes revealed the following: the major duplication of the key enzyme, PAL, 120 unigenes in leaves and 76 in flowers; the gene encoding C3&rsquo;H, 169 unigenes in leaves and 140 unigenes in flowers; duplicated unigenes of 4CL, 41 in leaves and 27 in flowers. In addition, C4H unigenes had 12 unigenes in the leaves of A. spathulifolius and four in the flowers. The characterization of the BAHDs superfamily members identified 82 in leaves and 72 in flowers. Among them, phylogenetic analysis showed that five unigenes encoded HQT and three en-coded HCT in A. spathulifolius. The three HQT are common to both leaves and flowers, whereas the two HQT were specialized for leaves. The pattern of HQT synthesis was upregulated in flowers, whereas HCT was expressed strongly in the leaves of A. spathulifolius. Overall, 4CL, C4H, and HQT are expressed strongly in flowers, and caffeic acid and HCT show more expression in leaves. Therefore, CQA biosynthesis occurs in the flowers of A. spathulifolius rather than leaves.

Characterization of mutant alleles of myospheroid, the gene encoding the beta subunit of the Drosophila PS integrins.

Genetics ◽  
1992 ◽  
Vol 132 (2) ◽  
pp. 519-528 ◽  
Author(s):  
T A Bunch ◽  
R Salatino ◽  
M C Engelsgjerd ◽  
L Mukai ◽  
R F West ◽  
...  
Keyword(s):  
Chromosome Pairing ◽  
Protein Product ◽  
Beta Subunit ◽  
Wild Type ◽  
Muscle Attachment ◽  
Negative Interaction ◽  
Gene Encoding ◽  
Attachment Sites ◽  
Muscle Attachment Sites

Abstract This paper presents the characterization of nine alleles of myospheroid, which encodes the beta PS subunit of the Drosophila PS integrins. On Southern blots, the mysXB87, mysXN101 and mysXR04 genes yield restriction digest patterns similar to that seen for wild-type chromosomes, however the mys1 and mysXG43 genes contain detectable deletions. mys1, mysXB87 and mysXG43 make little or no stable protein product, and genetically behave as strong lethal alleles. For the mysXN101 mutation, protein product is seen on immunoblots and a reduced amount of beta PS protein is seen at muscle attachment sites of embryos; this mutant protein retains some wild-type function, as revealed by complementation tests with weak alleles. Protein is also seen on immunoblots from mysXR04 embryos, and this allele behaves as an antimorph, being more deleterious in some crosses than the complete deficiency for the locus. mysts2 and mysnj42 are typically lethal in various combinations with other alleles at high temperatures only, but even at high physiological temperatures, neither appears to eliminate gene function completely. The complementation behaviors of mysts1 and mysts3 are quite unusual and suggest that these mutations involve regulatory phenomena. For mysts3, the data are most easily explained by postulating transvection effects at the locus. The results for mysts1 are less straightforward, but point to the possibility of a chromosome pairing-dependent negative interaction.

scholarly journals Release of Nonstop Ribosomes Is Essential

mBio ◽  
2014 ◽  
Vol 5 (6) ◽  
Author(s):  
Heather A. Feaga ◽  
Patrick H. Viollier ◽  
Kenneth C. Keiler
Keyword(s):  
Messenger Rna ◽  
Caulobacter Crescentus ◽  
Stop Codon ◽  
Selective Advantage ◽  
Gene Encoding ◽  
Content Type ◽  
Bacterial Phyla ◽  
Almost All

ABSTRACTBacterial ribosomes frequently translate to the 3′ end of an mRNA without terminating at a stop codon. Almost all bacteria use the transfer-messenger RNA (tmRNA)-basedtrans-translation pathway to release these “nonstop” ribosomes and maintain protein synthesis capacity.trans-translation is essential in some species, but in others, such asCaulobacter crescentus,trans-translation can be inactivated. To determine whytrans-translation is dispensable inC. crescentus, a Tn-seq screen was used to identify genes that specifically alter growth in cells lackingssrA, the gene encoding tmRNA. One of these genes,CC1214, was essential in ΔssrAcells. Purified CC1214 protein could release nonstop ribosomesin vitro. CC1214 is a homolog of theEscherichia coliArfB protein, and using the CC1214 sequence, ArfB homologs were identified in the majority of bacterial phyla. Most species in whichssrAhas been deleted contain an ArfB homolog, suggesting that release of nonstop ribosomes may be essential in most or all bacteria.IMPORTANCEGenes that are conserved across large phylogenetic distances are expected to confer a selective advantage. The genes required fortrans-translation,ssrAandsmpB, have been found in >99% of sequenced bacterial genomes, suggesting that they are broadly important. However, these genes can be deleted in some species without loss of viability. The identification and characterization ofC. crescentusArfB reveals whytrans-translation is not essential inC. crescentusand suggests that many other bacteria are likely to use ArfB to survive whentrans-translation is compromised.

scholarly journals Identification and Characterization of the Bacillus thuringiensis phaZ Gene, Encoding New Intracellular Poly-3-Hydroxybutyrate Depolymerase

2006 ◽  
Vol 188 (21) ◽  
pp. 7592-7599 ◽  
Author(s):  
Chi-Ling Tseng ◽  
Hui-Ju Chen ◽  
Gwo-Chyuan Shaw
Keyword(s):  
Bacillus Thuringiensis ◽  
Wild Type ◽  
Gene Encoding ◽  
Significant Similarity ◽  
Phb Depolymerase ◽  
New Type ◽  
Identification And Characterization

ABSTRACTA gene that codes for a novel intracellular poly-3-hydroxybutyrate (PHB) depolymerase has now been identified in the genome ofBacillus thuringiensissubsp.israelensisATCC 35646. This gene, previously annotated as a hypothetical 3-oxoadipate enol-lactonase (PcaD) gene and now designatedphaZ, encodes a protein that shows no significant similarity with any known PHB depolymerase. Purified His-tagged PhaZ could efficiently degrade trypsin-activated native PHB granules as well as artificial amorphous PHB granules and release 3-hydroxybutyrate monomer as a hydrolytic product, but it could not hydrolyze denatured semicrystalline PHB. In contrast, purified His-tagged PcaD ofPseudomonas putidawas unable to degrade trypsin-activated native PHB granules and artificial amorphous PHB granules. TheB. thuringiensisPhaZ was inactive againstp-nitrophenylpalmitate, tributyrin, and triolein. Sonication supernatants of the wild-typeB. thuringiensiscells exhibited a PHB-hydrolyzing activity in vitro, whereas those prepared from aphaZmutant lost this activity. ThephaZmutant showed a higher PHB content than the wild type at late stationary phase of growth in a nutrient-rich medium, indicating that this PhaZ can function as a PHB depolymerase in vivo. PhaZ contains a lipase box-like sequence (G-W-S102-M-G) but lacks a signal peptide. A purified His-tagged S102A variant had lost the PHB-hydrolyzing activity. Taken together, these results indicate thatB. thuringiensisharbors a new type of intracellular PHB depolymerase.

scholarly journals Characterization of the norB Gene, Encoding Nitric Oxide Reductase, in the Nondenitrifying Cyanobacterium Synechocystis sp. Strain PCC6803

2002 ◽  
Vol 68 (2) ◽  
pp. 668-672 ◽  
Author(s):  
Andrea B�sch ◽  
B�rbel Friedrich ◽  
Rainer Cramm
Keyword(s):  
Nitric Oxide ◽  
Reductase Activity ◽  
Nitric Oxide Reductase ◽  
Wild Type ◽  
Gene Encoding ◽  
Single Subunit ◽  
Transcriptional Fusions ◽  
Synechocystis Sp ◽  
Negative Mutant

ABSTRACT A norB gene encoding a putative nitric oxide reductase is present in the genome of the nondenitrifying cyanobacterium Synechocystis sp. strain PCC6803. The gene product belongs to the quinol-oxidizing single-subunit class of nitric oxide reductases, discovered recently in the denitrifier Ralstonia eutropha. Heterologous complementation of a nitric oxide reductase-negative mutant of R. eutropha with norB from Synechocystis restored nitric oxide reductase activity. With reduced menadione as the electron donor, an enzymatic activity of 101 nmol of NO per min per mg of protein was obtained with membrane fractions of Synechocystis wild-type cells. Virtually no nitric oxide reductase activity was present in a norB-negative mutant of Synechocystis. Growing cells of this mutant are more sensitive toward NO than wild-type cells, indicating that the presence of a nitric oxide reductase is beneficial for Synechocystis when the cells are exposed to NO. Transcriptional fusions with the chloramphenicol acetyltransferase reporter gene were constructed to monitor norB expression in Synechocystis. Transcription of norB was not enhanced by the addition of the NO-generating agent sodium nitroprusside.

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